Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.1.2.13 (aldolase)
3,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) homotetramer has been destabilized by site-directed mutagenesis at the two different subunit interfaces. A double mutant aldolase, Q125D/E224A, sediments as two distinct species, characteristic of a slow equilibrium, with velocities expected for the monomer and tetramer. The aldolase monomer is shown to be catalytically active following isolation from sucrose density gradients. The isolated aldolase monomer had 72% of the specific activity of the wild-type enzyme and a slightly lower Michaelis constant, clearly indicating that the quaternary structure is not required for catalysis. Cross-linking of the isolated monomer confirmed that it does not rapidly reequilibrate with the tetramer following isolation. There was a substantial difference between the tetramer and monomer in their inactivation by urea. The stability toward both urea and thermal inactivation of these oligomeric variants suggests a role for the quaternary structure in maintaining the stability of aldolase, which may be an important role of quaternary structure in many proteins.
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PMID:Disruption of the aldolase A tetramer into catalytically active monomers. 864 82

Considerations of the effect of a small cosolute on the sedimentation equilibrium distribution for a noninteracting protein have led to the development of a simple procedure for evaluating both the molecular weight of the protein and the second virial coefficient describing the excluded volume interaction between protein and cosolute. Its application is illustrated by analysis of sedimentation equilibrium distributions for bovine thyroglobulin and horse liver alcohol dehydrogenase in the presence of a range of sucrose concentrations, and also of those for aldolase in the presence of urea to obtain the subunit molecular weight of this tetrameric enzyme. The effects of sucrose concentration on the sedimentation coefficients of thyroglobulin, catalase, and horse liver alcohol dehydrogenase are also examined to demonstrate that the second virial coefficients for protein-cosolute excluded volume interaction may be determined, albeit with less precision, from the cosolute concentration required to render the sedimentation coefficient zero by virtue of its effect on the buoyancy term. These findings serve to reinforce the fact that the effects of small cosolutes usually ascribed to changes in "protein solvation" are envisaged more realistically in terms of excluded volume.
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PMID:Thermodynamic analysis of the effects of small inert cosolutes in the ultracentrifugation of noninteracting proteins. 885 55

Some cysteine-containing proteins upon sulfitolysis have been found to show anomalously retarded SDS-PAGE mobilities in non-reducing gels. These proteins include bovine serum albumin, ovalbumin, aldolase, ribonuclease and a recombinant fusion protein (XA) consisting of a portion of gamma-interferon linked to the A chain of human insulin. This mobility shift has been employed to determine the stability of the sulfonated products and to study the kinetics of the sulfitolysis reaction. Partially sulfonated products of intermediate shifts were observed at 0.01% beta-ME, while 0.05% beta-ME gave a shift characteristic of the completely reduced protein. The undiluted sulfitolysis reagent reacted with XA to give within 1 min a gel shift characteristic of the fully sulfitolysed protein. Its transition stages could be visualized at 15, 30 and 60 min when the reagent was diluted four-fold. In the presence of 8 M urea, the sulfitolysis of BSA was nearly complete at 30 min when the sulfitolysis reagent was used at a dilution of 1:5. However, under the same conditions BSA was predominantly unsulfitolysed in the absence of urea. In order to elucidate the mechanism of sulfonation shift, several derivatives of XA, e.g. performic acid oxidized, alkylated with (a) iodoacetamide and (b) iodoacetate, have been prepared. While the mobility of XASSO3- was sensitive to the presence of beta-ME, all other derivatives moved in a beta-ME-insensitive fashion. Furthermore, while the nonreducing mobilities of the acidic derivatives (-SSO3-, -SO3- and -SCH2CO2-) were anomalously retarded and identical, the mobility of the iodoacetamide derivative was intermediate between the retarded acidic derivatives above and XA below. These studies have suggested a role of the extended conformation of the A chain of insulin in causing a mobility shift of the acidic derivatives in this series. Similar results were observed in an analogous series of derivatives prepared from BSA. Non-denaturing gel filtration analyses of native vs. sulfitolysed samples of serum albumin, ovalbumin and ribonuclease have indicated that the sulfitolysed proteins elute earlier than their native counterparts and appear to be significantly larger than their true molecular weights. Circular dichroism analysis has indicated significant loss in helicity of sulfitolysed BSA. This suggests that the retarded mobility of sulfitolysed proteins seen on SDS-PAGE is likely to be due to an expansion in the hydrodynamic volumes of these proteins, a phenomenon triggered by cleavage of disulfide bonds and further accentuated by the introduction of strongly negatively charged sulfonates.
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PMID:Anomalous mobility of sulfitolysed proteins in SDS-PAGE. Analysis and applications. 889 91

Three unfolding domains in rabbit muscle aldolase destabilized in 3 M urea have been identified from their unfolding rate constants (0.10, 0.036, and 0.0064 min-1). The populations of folded and various, partially unfolded forms were determined by amide hydrogen exchange and mass spectrometry. Results of this study show that unfolding domains may include multiple, noncontiguous segments of the backbone and that different regions of helices may belong to different unfolding domains. In addition, these results show that the domain unfolding most rapidly is located distant from the subunit binding surfaces and has the greatest access to the denaturant. The bimodal intermolecular distributions of deuterium found in this study show that unfolding of these domains is cooperative. It is proposed that these unfolding domains are correlated with local energy minima in the free-energy folding surface of aldolase. In addition to the three unfolding domains, there are three short segments that do not unfold in 3 M urea. These segments, which are located in the subunit binding surface, identify the most stable regions of aldolase. This study also demonstrates that it is now possible to identify and characterize unfolding domains in relatively large (Mr 158 000) proteins.
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PMID:Identification of unfolding domains in large proteins by their unfolding rates. 957 40

Proteins with affinity to heparin under physiological conditions were isolated from bovine cerebral cortex. First, the extract of cerebral cortex was applied to a chondroitin polysulfate column under physiological conditions. Then, the pass-through fraction was applied to a heparin column. Among the bands on SDS polyacrylamide gel electrophoresis of the fraction bound to the heparin column, the major one was identified as fructose 1,6-bisphosphate aldolase (FPA), a cytosolic enzyme involved in the glycolytic pathway. The results indicated that FPA is a heparin-binding protein which exhibits no affinity to chondroitin polysulfate. The results of affinity chromatographies revealed that FPA binds to intact heparin and modified heparins desulfated at C2 OH of the iduronic acid residue or at C6 OH or C2 NH2 of the glucosamine residue. When 6-O-desulfated heparin was employed as the affinity ligand, a single peak having FPA activity was isolated from the extract of bovine cerebral cortex. By further Mono Q chromatography and Superdex gel-filtration, five isoenzymes were purified with more than 50% recovery. These isoenzymes were identified as FPA A4, A3C1, A2C2, A1C3, and C4 by native electrophoresis with and without 4 M urea and subsequent amino acid sequence analysis. The use of 6-O-desulfated heparin affinity chromatography thus facilitated the purification of FPA.
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PMID:Fructose 1,6-bisphosphate aldolase is a heparin-binding protein. 1005 44

In contrast to the rigid structures portrayed by X-ray diffraction, proteins in solution display constant motion which leads to populations that are momentarily unfolded. To begin to understand protein dynamics, we must have experimental methods for determining rates of folding and unfolding, as well as for identifying structures of folding and unfolding intermediates. Amide hydrogen exchange has become an important tool for such measurements. When urea is used to stabilize unfolded forms of proteins, the refolding rates may become slower than the rates of isotope exchange. In such cases, the intermolecular distribution of deuterium among the entire population of molecules may become bimodal, giving rise to a bimodal distribution of isotope peaks in mass spectra of the protein or its peptic fragments. When the protein is exposed continuously to D2O, the relative intensities of the two envelopes of isotope peaks give an integrated account of populations participating in the folding/unfolding process. However, when the protein is exposed only briefly to D2O, the relative intensities of the two envelopes of isotope peaks give an instantaneous measure of the folded/unfolded populations. Application of these two labeling methods to a large protein, aldolase, is described along with a discussion of specific parameters required to optimize these experiments.
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PMID:Comparison of continuous and pulsed labeling amide hydrogen exchange/mass spectrometry for studies of protein dynamics. 1043 6

Rabbit muscle aldolase is a homotetramer in which the subunits have a classical alpha/beta-barrel structure and Mr 39,212 Da. We have previously reported that aldolase incubated in 3 M urea has three unfolding domains distinguished by their different unfolding rates. The unfolding rates of these domains were determined from isotope patterns in the mass spectra of peptic fragments derived from aldolase incubated in 3 M urea and pulse labeled in (2)H2O. The present study extends this investigation to more thoroughly characterize the structures of these unfolding intermediates. Mass spectra of intact monomers had four envelopes of isotope peaks corresponding to four structural forms of aldolase. Analysis of the present results suggests that these structural forms consist of native aldolase and three forms that have one to three domains unfolded. The molecular masses of these four structural forms indicate that there are 107 residues in each of the three unfolding domains of aldolase. Present results also show that aldolase remains a tetramer in 4 M urea, even though hydrogen exchange and circular dichroism indicate that it has lost most of its secondary and tertiary structure. The abundances of unfolded domains, which were determined from mass spectra of either intact aldolase or its peptic fragments, were used to determine the abundances of specific, partially unfolded forms of aldolase. Kinetic modeling of the abundances of these structures suggests that these structures are formed sequentially as aldolase unfolds in urea.
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PMID:Hydrogen exchange demonstrates three domains in aldolase unfold sequentially. 1055 43

The proposed laboratory investigation was designed to evaluate the effects of acute exposure to both continuous and intermittent magnetic fields (MFs) (50 Hz-10 microT) on the circadian rhythm of clinical chemistry variables in humans: electrolytes (magnesium, calcium, phosphorus, sodium, potassium, and chloride), enzymes (amylase, lipase, aldolase, gamma glutamyl-transferase [GGT], lactate dehydrogenase [LDH], aspartate aminotransferase [ASAT], and alkaline phosphatase [ALP]), lipids (cholesterol, high-density lipoprotein [HDL], apolipoprotein A1 [ApoA1], and ApoB), proteins (total proteins and albumin), nitrogen substances (uric acid, urea, and creatinine), iron, glycemia, and transferrin. Young volunteers (32 subjects; 16 exposed and 16 sham exposed) were selected according to the screening criteria. Each subject participated in two sessions held within a 4-week period. In the first session, one group of volunteers (16 subjects) was exposed to a continuous MF and then, in the second session, to an intermittent MF. The second group (16 subjects) served as a control for both sessions. At each session, blood samples were collected at 3 h intervals from 11:00 to 20:00 and hourly from 22:00 to 08:00. The results indicate that both continuous and intermittent 50-Hz linearly polarized MFs of 10 microT intensity have no effects on the circadian rhythms or on the levels of the variables studied here.
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PMID:Assessment of the effects of nocturnal exposure to 50-Hz magnetic fields on the human circadian system. A comprehensive study of biochemical variables. 1058 79

Studies of protein unfolding and refolding may help us understand the more general problem of protein folding. Recent studies from this laboratory demonstrated that the unfolding and refolding of a large protein, rabbit muscle aldolase (M(r) 157 kDa), can be studied by combining amide hydrogen exchange and mass spectrometry. Results of these studies indicated that aldolase has three unfolding domains which likely unfold sequentially. Urea was used to increase the populations of partially unfolded states which were labeled with deuterium following a brief exposure to D(2)O. Electrospray ionization mass spectra of both the intact protein and its peptic fragments had multiple envelopes of isotope peaks from which the populations of unfolded forms were determined. The present study extends the previous investigations to include different urea concentrations and kinetic modeling of data taken as the system approaches equilibrium. Analysis of these results gives rate and equilibrium constants describing the unfolding and refolding processes characteristic of aldolase destabilized in urea. The change in solvent-accessible surface, which has been used as a reaction coordinate for protein folding, is estimated from the dependence of the equilibrium and rate constants on the concentration of urea.
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PMID:Rate and equilibrium constants for protein unfolding and refolding determined by hydrogen exchange-mass spectrometry. 1060 37

Cysteine residues and disulfide bonds are important for protein structure and function. We have developed a simple and sensitive method for determining the presence of free cysteine (Cys) residues and disulfide bonded Cys residues in proteins (<100 pmol) by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with protein database searching using the program Sequest. Free Cys residues in a protein were labeled with PEO-maleimide biotin immediately followed by denaturation with 8 M urea. Subsequently, the protein was digested with trypsin or chymotrypsin and the resulting products were analyzed by capillary LC/ESI-MS/MS for peptides containing modified Cys and/or disulfide bonded Cys residues. Although the MS method for identifying disulfide bonds has been routinely employed, methods to prevent thiol-disulfide exchange have not been well documented. Our protocol was found to minimize the occurrence of the thiol-disulfide exchange reaction. The method was validated using well-characterized proteins such as aldolase, ovalbumin, and beta-lactoglobulin A. We also applied this method to characterize Cys residues and disulfide bonds of beta 1,4-galactosyltransferase (five Cys), and human blood group A and B glycosyltransferases (four Cys). Our results demonstrate that beta 1,4-galactosyltransferase contains one free Cys residue and two disulfide bonds, which is in contrast to work previously reported using chemical methods for the characterization of free Cys residues, but is consistent with recently published results from x-ray crystallography. In contrast to the results obtained for beta 1,4-galactosyltransferase, none of the Cys residues in A and B glycosyltransferases were found to be involved in disulfide bonds.
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PMID:Characterization of cysteine residues and disulfide bonds in proteins by liquid chromatography/electrospray ionization tandem mass spectrometry. 1097 99


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